Sadie C. Slater

Dismantling of Cadherin-Mediated Cell-Cell Contacts Modulates Smooth Muscle Cell Proliferation

Abstract— Proliferation of vascular smooth muscle cells (VSMCs) contributes to intimal thickening during atherosclerosis and restenosis. The cadherins are transmembrane proteins, which form cell-cell contacts and may regulate VSMC proliferation. In this study, N-cadherin protein concentration was significantly reduced by stimulation of proliferation with fetal calf serum (FCS) and platelet-derived growth factor-BB (PDGF-BB) in human saphenous vein VSMCs. Furthermore, overexpression of a truncated N-cadherin, which acts as a dominant-negative increased VSMC proliferation. The amount of an extracellular fragment of N-cadherin (≈90 kDa) in the media after 24 hours was increased by 12-fold by FCS and 11-fold by PDGF-BB, suggesting that N-cadherin levels are regulated by proteolytic shedding. Incubation with a synthetic metalloproteinase inhibitor or adenoviral overexpression of the endogenous tissue inhibitors of metalloproteinases (TIMPs) demonstrated that metalloproteinase activity was responsible in part for this proteolysis. Although total levels of &bgr;-catenin protein were not affected, &bgr;-catenin was translocated to the nucleus after stimulation with FCS and PDGF-BB. Our data indicates cadherin-mediated cell-cell contacts modulate proliferation in VSMCs. Furthermore, disruption of N-cadherin cell-cell contacts mediated in part by metalloproteinase activity occurs during VSMC proliferation, releasing &bgr;-catenin and possibly inducing &bgr;-catenin-mediated intracellular signaling.

Regulation of Smooth Muscle Cell Proliferation by β-Catenin/T-Cell Factor Signaling Involves Modulation of Cyclin D1 and p21 Expression

We previously observed that stimulation of vascular smooth muscle cell (VSMC) proliferation with growth factors is associated with dismantling of cadherin junctions and nuclear translocation of &bgr;-catenin. In this study we demonstrate directly that growth factors stimulate &bgr;-catenin/T-cell factor (TCF) signaling in primary VSMCs. To determine whether &bgr;-catenin/TCF signaling regulates VSMC proliferation via modulation of the &bgr;-catenin/TCF responsive cell cycle genes, cyclin D1 and p21, we inhibited &bgr;-catenin/TCF signaling by adenoviral-mediated over-expression of N-Cadherin, ICAT (an endogenous inhibitor of &bgr;-catenin/TCF signaling), or a dominant negative (dn) mutant of TCF-4. N-cadherin, ICAT or dnTCF-4 over-expression significantly reduced proliferation of isolated human VSMCs by approximately 55%, 80%, and 45% respectively. Similar effects were observed in human saphenous vein medial segments where proliferation was reduced by approximately 55%. Transfection of dnTCF-4 in the ISS10 human VSMC line significantly lowered TCF and cyclin D1 reporter activity but significantly elevated p21 reporter activity, indicating regulation of these genes by &bgr;-catenin/TCF signaling. In support of this, over-expression of N-cadherin, ICAT or dnTCF-4 in isolated human VSMCs significantly lowered levels of cyclin D1 mRNA and protein levels. In contrast, over-expression of N-Cadherin, ICAT or dnTCF4 significantly elevated p21 mRNA and protein levels. In summary, we have demonstrated that increasing N-cadherin and inhibiting &bgr;-catenin/TCF signaling reduces VSMC proliferation, decreases the expression of cyclin D1 and increases levels of the cell cycle inhibitor, p21. We therefore suggest that the N-cadherin and &bgr;-catenin/TCF signaling pathway is a key modulator of VSMC proliferation via regulation of these 2 &bgr;-catenin/TCF responsive genes.

MMP-9 and -12 cause N-cadherin Shedding, and thereby β-catenin Signalling and Vascular Smooth Muscle Cell Proliferation

AIMS Vascular smooth muscle cell (VSMC) proliferation contributes to intimal thickening in restenosis and atherosclerosis. Previously, we demonstrated that matrix-degrading metalloproteinase (MMP)-dependent shedding of the extracellular portion of N-cadherin increased VSMC proliferation via elevation of beta-catenin signalling and cyclin D1 expression. In this study, we aimed to determine whether MMP-2, -9, -12, or -14 regulates VSMC proliferation via N-cadherin shedding. METHODS AND RESULTS N-cadherin shedding was significantly impaired in proliferating mouse aortic VSMCs deficient in MMP-9 (MMP-9(-/-)) and MMP-12 (MMP-12(-/-)) compared with wild-type controls (1.1 +/- 0.7- and 1.0 +/- 0.1- vs. 2.0 +/- 0.2-fold). Furthermore, proliferating VSMCs subjected to MMP-9 or -12 siRNA knockdown or deficient in MMP-9 or -12 showed significantly increased cellular levels of N-cadherin compared with controls (1.7 +/- 0.2-, 2.7 +/- 0.6-, and 3.5 +/- 1.6-, 1.7 +/- 0.2-fold, respectively). Incubation of VSMCs with active MMP-9 or -12 independently increased N-cadherin cleavage. Additionally, beta-catenin signalling was significantly reduced by 52 +/- 17 and 81 +/- 12% in MMP-9(-/-) and -12(-/-) proliferating VSMCs, respectively, and this was corroborated by siRNA knockdown of MMP-9 and -12. Decreased beta-catenin signalling coincided with significantly reduced proliferation and cyclin D1 protein levels in MMP-9(-/-) and -12(-/-) cells. Little or no additive effect was observed with combined modulation of MMP-9 and -12 in all experiments. In contrast, N-cadherin shedding and VSMC proliferation were not modulated by MMP-2 and -14. CONCLUSION In conclusion, we propose that MMP-9 and -12 promote intimal thickening by independent cleavage of N-cadherin, which elevates VSMC proliferation via beta-catenin signalling.

R-Cadherin:β-Catenin Complex and Its Association With Vascular Smooth Muscle Cell Proliferation

Objective—Vascular smooth muscle cell (VSMC) proliferation is an important component of atherosclerosis, restenosis after angioplasty and stent placement, and vein graft failure. Outside-in signaling from the cadherin:β-catenin complex can increase transcription of the cell–cycle gene cyclin D1; however, its role in VSMC proliferation has only recently been considered. Methods and Results—We examined the involvement of R-cadherin and β-catenin in VSMC proliferation in balloon-injured carotid arteries in vivo and aortic rings in vitro. The number of medial VSMCs positive for R-cadherin was significantly reduced by 32%±5%, 52%±10%, and 23%±2% at 0.25, 24, and 48 hours after injury in vivo, respectively. These changes in cadherin expression coincided with the detection of nuclear β-catenin and elevated cyclin D1 expression. Furthermore, loss of R-cadherin expression was associated with medial VSMC proliferation. Inhibition of classical cadherin function with a HAV peptide and R-cadherin neutralizing antibodies significantly increased proliferation by 4.3±1.0-fold and 4.1±0.98-fold, and increased the number of cells with β-catenin in the nucleus and expressing cyclin D1 in aortic rings. Conclusions—These results suggest that R-cadherin expression and β-catenin signaling may be associated with increased cyclin D1 expression and VSMC proliferation and may therefore play an important role in vascular disease.

N-Cadherin-Dependent Cell-Cell Contacts Promote Human Saphenous Vein Smooth Muscle Cell Survival

Objective—Vascular smooth muscle cell (VSMC) apoptosis is thought to contribute to atherosclerotic plaque instability. Cadherin mediates calcium-dependent homophilic cell–cell contact. We studied the role of N-cadherin in VSMC apoptosis. Methods and Results—Human saphenous vein VSMCs were grown in agarose-coated wells to allow cadherin-mediated aggregate formation. Cell death and apoptosis were determined after disruption of cadherins using several approaches (n≥3 per approach). Calcium removal from culture medium or addition of nonspecific cadherin antagonist peptides significantly decreased aggregate formation and increased cell death by apoptosis (34±6% versus 75±1% and 19±1% versus 40±5%, respectively; P<0.05). Specific inhibition of N-cadherin using antagonists and neutralizing antibodies similarly increased apoptosis. Supporting this, overexpression of full-length N-cadherin significantly reduced VSMC apoptosis from 44±10% to 20±3% (P<0.05), whereas abolishing N-cadherin expression by overexpression of a dominant-negative N-cadherin significantly, even in the presence of cell–matrix contacts, increased apoptosis from 9±2% to 50±1% (P<0.05). Interestingly, cell–cell contacts provided a similar degree of protection from apoptosis to cell–matrix contacts. Finally, N-cadherin–mediated cell–cell contacts initiated anti-apoptotic signaling by increasing Akt and Bad phosphorylation. Conclusions—Our results indicate that VSMC survival is dependent on N-cadherin–mediated cell–cell contacts, which could be important in the context of plaque instability.

Chronic exposure to laminar shear stress induces Kruppel-like factor 2 in glomerular endothelial cells and modulates interactions with co-cultured podocytes

Laminar shear stress (LSS), induced by flowing blood, plays a key role in determining vascular health by modulating endothelial behaviour and vascular tone. In systemic endothelium many of the beneficial effects of chronic LSS are mediated through the transcription factor Kruppel-like factor 2 (KLF2), but little is known regarding the role of chronic LSS in the renal glomerulus. We demonstrate that exposure of glomerular endothelial cells to chronic (>24h) LSS of 10 dyn/cm(2) increases phosphorylation of extra-cellular signal-related kinase 5 (ERK5) and increases expression of KLF2, leading to increased expression of the downstream molecules endothelial nitric oxide synthase (eNOS), thrombomodulin, endothelin-1 and nitric oxide. However, the proportion of eNOS which was phosphorylated at serine 1117 and threonine 495 residues was decreased. We demonstrated dependence of these effects on the ERK5 pathway by using the inhibitor UO126. We found high levels of KLF2 expression in human glomeruli confirming the relevance of our in vitro observations and, as KLF2 is specifically induced by chronic LSS, suggesting the physiological importance of shear stress in the glomerulus. Conditioned medium from glomerular endothelial cells under chronic LSS decreased podocyte monolayer resistance and increased phosphorylation of vasodilator-stimulated phosphoprotein. The latter effect was more pronounced using a novel insert-based direct co-culture system in which endothelial cells were exposed to chronic LSS. These data provide the first direct evidence of glomerular endothelial cell to podocyte cross-talk.

An In Vitro Model of the Glomerular Capillary Wall Using Electrospun Collagen Nanofibres in a Bioartificial Composite Basement Membrane

The filtering unit of the kidney, the glomerulus, contains capillaries whose walls function as a biological sieve, the glomerular filtration barrier. This comprises layers of two specialised cells, glomerular endothelial cells (GEnC) and podocytes, separated by a basement membrane. Glomerular filtration barrier function, and dysfunction in disease, remains incompletely understood, partly due to difficulties in studying the relevant cell types in vitro. We have addressed this by generation of unique conditionally immortalised human GEnC and podocytes. However, because the glomerular filtration barrier functions as a whole, it is necessary to develop three dimensional co-culture models to maximise the benefit of the availability of these cells. Here we have developed the first two tri-layer models of the glomerular capillary wall. The first is based on tissue culture inserts and provides evidence of cell-cell interaction via soluble mediators. In the second model the synthetic support of the tissue culture insert is replaced with a novel composite bioartificial membrane. This consists of a nanofibre membrane containing collagen I, electrospun directly onto a micro-photoelectroformed fine nickel supporting mesh. GEnC and podocytes grew in monolayers on either side of the insert support or the novel membrane to form a tri-layer model recapitulating the human glomerular capillary in vitro. These models will advance the study of both the physiology of normal glomerular filtration and of its disruption in glomerular disease.

Vascular Endothelial Growth Factor-C, a Potential Paracrine Regulator of Glomerular Permeability, Increases Glomerular Endothelial Cell Monolayer Integrity and Intracellular Calcium

We have previously reported expression of vascular endothelial growth factor (VEGF)-A and -C in glomerular podocytes and actions of VEGF-A on glomerular endothelial cells (GEnC) that express VEGF receptor-2 (VEGFR-2). Here we define VEGFR-3 expression in GEnC and investigate the effects of the ligand VEGF-C. Renal cortex and cultured GEnC were examined by microscopy, and both cell and glomerular lysates were assessed by Western blotting. VEGF-C effects on trans-endothelial electrical resistance and albumin flux across GEnC monolayers were measured. The effects of VEGF-C156S, a VEGFR-3-specific agonist, and VEGF-A were also studied. VEGF-C effects on intracellular calcium ([Ca2+]i) were measured using a fluorescence technique, receptor phosphorylation was examined by immunoprecipitation assays, and phosphorylation of myosin light chain-2 and VE-cadherin was assessed by blotting with phospho-specific antibodies. GEnC expressed VEGFR-3 in tissue sections and culture, and VEGF-C increased trans-endothelial electrical resistance in a dose-dependent manner with a maximal effect at 120 minutes of 6.8 Omega whereas VEGF-C156S had no effect. VEGF-C reduced labeled albumin flux by 32.8%. VEGF-C and VEGF-A increased [Ca2+]i by 15% and 39%, respectively. VEGF-C phosphorylated VEGFR-2 but not VEGFR-3, myosin light chain-2, or VE-cadherin. VEGF-C increased GEnC monolayer integrity and increased [Ca2+]i, which may be related to VEGF-C-S particular receptor binding and phosphorylation induction characteristics. These observations suggest that podocytes direct GEnC behavior through both VEGF-C and VEGF-A.

Epigenetic Profile of Human Adventitial Progenitor Cells Correlates With Therapeutic Outcomes in a Mouse Model of Limb Ischemia

Objective— We investigated the association between the functional, epigenetic, and expressional profile of human adventitial progenitor cells (APCs) and therapeutic activity in a model of limb ischemia. Approach and Results— Antigenic and functional features were analyzed throughout passaging in 15 saphenous vein (SV)–derived APC lines, of which 10 from SV leftovers of coronary artery bypass graft surgery and 5 from varicose SV removal. Moreover, 5 SV-APC lines were transplanted (8×105 cells, IM) in mice with limb ischemia. Blood flow and capillary and arteriole density were correlated with functional characteristics and DNA methylation/expressional markers of transplanted cells. We report successful expansion of tested lines, which reached the therapeutic target of 30 to 50 million cells in ≈10 weeks. Typical antigenic profile, viability, and migratory and proangiogenic activities were conserved through passaging, with low levels of replicative senescence. In vivo, SV-APC transplantation improved blood flow recovery and revascularization of ischemic limbs. Whole genome screening showed an association between DNA methylation at the promoter or gene body level and microvascular density and to a lesser extent with blood flow recovery. Expressional studies highlighted the implication of an angiogenic network centered on the vascular endothelial growth factor receptor as a predictor of microvascular outcomes. FLT-1 gene silencing in SV-APCs remarkably reduced their ability to form tubes in vitro and support tube formation by human umbilical vein endothelial cells, thus confirming the importance of this signaling in SV-APC angiogenic function. Conclusions— DNA methylation landscape illustrates different therapeutic activities of human APCs. Epigenetic screening may help identify determinants of therapeutic vasculogenesis in ischemic disease.

Expansion and Characterization of Neonatal Cardiac Pericytes Provides a Novel Cellular Option for Tissue Engineering in Congenital Heart Disease

Background Living grafts produced by combining autologous heart-resident stem/progenitor cells and tissue engineering could provide a new therapeutic option for definitive correction of congenital heart disease. The aim of the study was to investigate the antigenic profile, expansion/differentiation capacity, paracrine activity, and pro-angiogenic potential of cardiac pericytes and to assess their engrafting capacity in clinically certified prosthetic grafts. Methods and Results CD34pos cells, negative for the endothelial markers CD31 and CD146, were identified by immunohistochemistry in cardiac leftovers from infants and children undergoing palliative repair of congenital cardiac defects. Following isolation by immunomagnetic bead-sorting and culture on plastic in EGM-2 medium supplemented with growth factors and serum, CD34pos/CD31neg cells gave rise to a clonogenic, highly proliferative (>20 million at P5), spindle-shape cell population. The following populations were shown to expresses pericyte/mesenchymal and stemness markers. After exposure to differentiation media, the expanded cardiac pericytes acquired markers of vascular smooth muscle cells, but failed to differentiate into endothelial cells or cardiomyocytes. However, in Matrigel, cardiac pericytes form networks and enhance the network capacity of endothelial cells. Moreover, they produce collagen-1 and release chemo-attractants that stimulate the migration of c-Kitpos cardiac stem cells. Cardiac pericytes were then seeded onto clinically approved xenograft scaffolds and cultured in a bioreactor. After 3 weeks, fluorescent microscopy showed that cardiac pericytes had penetrated into and colonized the graft. Conclusions These findings open new avenues for cellular functionalization of prosthetic grafts to be applied in reconstructive surgery of congenital heart disease.